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  • C07D459/00—Heterocyclic compounds containing benz [g] indolo [2, 3-a] quinolizine ring systems, e.g. yohimbine; 16, 18-lactones thereof, e.g. reserpic acid lactone
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  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/47—Quinolines; Isoquinolines
  • A61K31/475—Quinolines; Isoquinolines having an indole ring, e.g. yohimbine, reserpine, strychnine, vinblastine
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  • A61K31/13—Amines
  • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
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  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/365—Lactones
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  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
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  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
  • C07D471/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains three hetero rings
  • C07D471/14—Ortho-condensed systems
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  • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
  • C07D487/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains three hetero rings
  • C07D487/14—Ortho-condensed systems
• • G—PHYSICS
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  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
• • G—PHYSICS
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  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value
  • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

• the invention relates to combinations of Rauwolfia alkaloid derivatives and a
• Anti-cancer therapy utilizes a combination of therapeutic interventions such as surgery, radiation therapy and chemotherapy.
• Surgery and radiation therapy are generally confined locally to the main site of tumor growth, while chemotherapy is applied to prevent tumor re-growth or against distant tumor foci.
• Chemotherapeutic agents are also used to reduce tumor growth to manage disease progression when radiotherapy or surgery is not an option.
• Immunosuppressive agents are clinically used to suppress a pathological immune reaction which targets the own body (autoimmunity) or overshooting immune reactions as seen in allergy. They are also used to treat transplant rejection caused by the immune system.
• Basic to immune responses is activation and proliferation of T cells following antigenic stimulation, which act in turn as helper cells for B cells, regulatory cells or effector cells.
• Immunosuppressive agents such as rapamycin or cyclosporine A act by inhibiting early T cell activation/proliferation. As both cancer and immune responses involve cell proliferation, some agents, for example rapamycin or its analogs, were initially used for immunosuppression but found later application as anticancer agents (Recher et al., Blood 2005, 105:2527-34).
• Chemotherapeutic drugs are most effectively used in combination therapy. The rationale is to apply drugs that work via different mechanisms in order to decrease the probability of developing drug-resistant cancer cells. Combination therapy also allows, for certain drug combinations, an optimal combined dose to minimize side effects. This is crucial as standard chemotherapeutic agents target essential cellular process such as DNA
• the Rauwolfia alkaloid derivatives of formula (I) are synthetic derivatives of reserpine, an anti-hypertensive and anti-psychotic agent (J.A.M.A., Vol. 170, Nr.17, Aug. 22, 1959, p. 2092). Reserpine and its derivatives like syrosingopine are rarely used today due to the development of better drugs with fewer side-effects. Reserpine acts by inhibition of the vesicular monoamine transporter leading to catecholamine depletion and this mode of action is believed to be shared by all the reserpine derivatives with an anti-hypertensive effect.
• Mitochondria contain the energy generating system of a cell, whereby electrons from metabolism pass through complexes I - IV of the electron transfer chain (ETC) leading to extrusion of protons from complex I, ⁇ and IV and to a reflux of protons through complex V with concomitant formation of chemical energy in the form of adenosine triphosphate (ATP).
• Oxygen serves as the ultimate electron acceptor and is reduced to H 2 0.
• Critical in this process is the inner mitochondrial membrane, as protons extruded from the complexes pass from the matrix through this membrane into the inter- membrane space, generating a positive membrane potential of 150-200 mV.
• Dyes such as TMRM (tetramethylrhodamine methyl ester) pass this membrane and accumulate in the mitochondrial matrix, whereby the intensity of the fluorescent signal depends on the strength of the membrane potential.
• TMRM tetramethylrhodamine methyl ester
• a number of well described agents inhibit mitochondrial function and may be regarded as mitochondriotoxic agents.
• So called uncoupling agents such as FCCP (carbonyl cyanide-p- trifluoromethoxyphenylhydrazone) uncouple the flow of protons from ATP synthesis, leading to a collapse of the membrane potential with resulting loss of ATP synthesis.
• Mitochondria are believed to be ancestrally engulfed bacteria. They contain a DNA genome encoding several components of the ETC, as well as components of the mitochondrial ribosome.
• Agents targeting the mitochondrial genome such as certain HIV-inhibitors of the class of nucleoside analogs, e.g. stavudine (D4T), are toxic for mitochondria as they ultimately destroy the ETC and the
• Metformin is a widely used biguanide drug for type 2 diabetes. It is related to buformin and phenformin, two biguanides not used anymore in diabetes therapy due to toxicity.
• the main clinical benefit of metformin in the treatment of type 2 diabetes is the suppression of hepatic gluconeogenesis to reduce hyperglycemia and improved insulin sensitivity; these effects are believed to be exerted by metformin-dependent stimulation of AMP-activated protein kinase (AMPK) activity.
• AMPK AMP-activated protein kinase
• Basic to this effect is the fact, that metformin and other biguanides inhibit complex I of the respiratory chain (electron transfer chain) of mitochondria (El-Mir et al., J Biol Chem 2000, 275:223-228).
• Rauwolfia alkaloid derivatives of formula (I) described hereinbelow are useful in the treatment of cancer or autoimmune diseases or in an immunosuppressive treatment when used in combination with mitochondrial inhibitors.
• Rl, R3 and R4 independently of one another represent: hydrogen, Q-C 3 alkyl, halogen, Q-C 3 alkoxy, amino, Q-C 3 alkylamino, Q-C 3 dialkylamino, or hydroxyl;
• R2 represents hydrogen, Q-C 3 alkyl, halogen, amino, Q-C 3 alkylamino, Q-C 3 dialkylamino, or a group -OR2a;
• R2a represents hydrogen, Q-C3 alkyl, formyl or an optionally substituted group selected from alkylcarbonyl, alkoxycarbonyl, arylcarbonyl and heteroarylcarbonyl;
• R5 represents C C 4 alkyl
• R6, R7, R8 and R9 independently of one another represent:
• G represents a group selected from:
• Al represents an optionally substituted group selected from alkyl, aryl, heteroaryl, cycloalkyl and heterocyclyl,
• A2 represents an optionally substituted group selected from alkyl, cycloalkyl, heterocyclyl, -(CH 2 ) n -aryl and -(CH 2 ) n -heteroaryl and
• n 0, 1, 2 or 3
• mitochondrial inhibitor for use in the treatment of cancer or autoimmune diseases or in an immunosuppressive treatment in combination with a compound of said formula (I).
• the invention therefore also relates to compounds of the above formula (I), wherein
• Rl, R3 and R4 independently of one another represent:
• R2 represents hydrogen, Q-C 3 alkyl, halogen, amino, Q-C 3 alkylamino, Q-C 3 dialkylamino, or a group -OR2a;
• R2a represents hydrogen, Q-C 3 alkyl, formyl or an optionally substituted group selected from alkylcarbonyl, alkoxycarbonyl, arylcarbonyl and heteroarylcarbonyl;
• R5 represents C C 4 alkyl
• R6, R7, R8 and R9 independently of one another represent:
• G represents a group selected from:
• Al represents an optionally substituted group selected from alkyl, aryl, heteroaryl, cycloalkyl and heterocyclyl,
• A2 represents an optionally substituted group selected from alkyl, cycloalkyl, heterocyclyl, -(CH 2 ) n -aryl and -(CH 2 ) n -heteroaryl and
• n 0, 1, 2 or 3
• R2 in formula (I) must not be hydrogen or methoxy when A 1 is an alkyl group.
• alkyl as used in this application includes in particular optionally substituted branched or unbranched alkyl groups having e.g. 1 to 10 carbon atoms, preferably Q-Cealkyl, more preferably Q- ⁇ alkyl, including e.g. methyl, ethyl, propyl (n-propyl or isopropyl), butyl (n-butyl, isobutyl or tertiary-butyl), pentyl (including n-pentyl and isopentyl) or branched and unbranched hexyl, heptyl or octyl residues.
• Q-Csalkyl is most preferred, in particular methyl and ethyl.
• cycloalkyl refers preferably to optionally substituted cycloalkyl groups having 3 to 12 ring atoms which may be arranged in one or more rings, more preferably to C3 -Qocycloalkyl like e.g. C 3 -C 7 cycloalkyl.
• Preferred specific examples of cycloalkyl moieties include optionally substituted cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl groups.
• heterocyclyl refers to non-aromatic ring systems having 3 to 10 ring atoms which may be arranged in one or more rings, wherein one, two, three or four of the carbon ring atoms are replaced by a heteroatom selected from heteroatoms such as N, O or S.
• Monocyclic C3-C 7 heterocyclyl groups are particularly preferred, including, but not limited to morpholyl, thiomorpholyl, piperazyl, piperidyl, tetrahydrofuryl, pyrrolidyl, oxazolyl, lH-pyrazolyl, lH-tetrazolyl and the like.
• heterocycloalkyl groups are optionally substituted as described below. They can also include one or more unsaturated bond, in particular doublebond like e.g. 2H-pyrrol, 4H- imidazol, 4-H-pyrazol, 4H-oxazol or 4H-isooxazol or 2H- or 4H-pyran.
• aryl means a carbocyclic aromatic group having 6 to 14, preferably 6 to 10 ring atoms.
• aryl groups are phenyl, naphthyl and the like.
• a particularly preferred example of aryl is phenyl.
• the aryl group described above may be substituted independently with one, two, or three substituents, preferably one or two substituents as described below.
• heteroaryl means a monocyclic or bicyclic radical of 5 to 14, preferably 5 to 10, more preferably 5 or 6, ring atoms with at least one aromatic ring containing one, two, or three ring heteroatoms selected from N, O, and S, the remaining ring atoms being C.
• One or two ring carbon atoms of the heteroaryl group may optionally be replaced with a carbonyl group.
• heteroaryl are pyridyl, pyrazinyl, pyrimidyl, thiophenyl, oxadiazolyl, pyrazolyl, oxazolyl, triazolyl, tetrazolyl, 1,8-naphthyridinyl, quinoxalinyl, quinazolinyl, indolizinyl, phenantridinyl, phenothiazinyl or phenoxazinyl and the like.
• the heteroaryl group described above may optionally be substituted with one, two, or three substituents, preferably one or two substituents as described below.
• Preferred optional substituents of the aforementioned alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl groups include halogen, in particular fluoro, chloro, bromo and iodo, hydroxyl, alkoxy, in particular Q-C 3 alkoxy, amino, Q-C 3 alkylamino, Q-C 3
• dialkylamino, cyano and carboxy and the like are preferred substituents.
• alkyl substituents in particular Q-CsaLkyl
• haloalkyl substituents in particular halo C - C 3 alkyl substituents, like e.g. trifluoromethyl.
• Particularly preferred compounds of formula (I) are on one hand the compounds wherein G represents a group selected from:
• Al represents an optionally substituted group selected from aryl, heteroaryl, cycloalkyl and heterocyclyl,
• A2 represents an optionally substituted group selected from alkyl, cycloalkyl, heterocyclyl, -(CH 2 ) n -aryl and -(CH 2 ) n -heteroaryl and
• n 0, 1, 2 or 3.
• Rl, R2, R3 and R4 in said compounds are preferably hydrogen, Q-C 3 alkyl, halogen, C - C 3 alkoxy, amino, Q-C 3 alkylamino, Q-C 3 dialkylamino. More preferably Rl, R3, R4 are hydrogen; and R2 is hydrogen or methoxy. Most preferred are the compounds of this embodiment wherein Rl, R3 and R4 are hydrogen and R2 is methoxy.
• R5 in said compounds is preferably C C 4 alkyl, in particular methyl
• R6, R7, R8 and R9 are preferably selected from hydrogen, Q-C 3 alkyl, halogen, Q-C 3 alkoxy, amino, Q-C 3 alkylamino, Q-C 3 dialkylamino and hydroxyl, more preferably from hydrogen, halogen, Q-C 3 alkoxy and hydroxyl.
• R6, and R9 are hydrogen and R7 and R8 are independently hydrogen or methoxy.
• G in said compounds is preferably a group -OC0 2 (CH 2 ) n -Al, with n being 0 or 1 and Al optionally substituted aryl, heteroaryl, in particular pyridyl or phenyl, optionally substituted with halogen or methoxy; or
• A2 being optionally substituted alkyl, -(CH 2 ) n -aryl, -(CH 2 ) n -heteroaryl, in particular CrC 4 alkyl, with n being 0 or 1.
• R2 represents a group -OR2a; and R2a represents hydrogen, formyl or an optionally substituted group selected from alkylcarbonyl, alkoxycarbonyl, arylcarbonyl and heteroarylcarbonyl, the optional substituents being e.g. selected from halogen, hydroxyl, Q-C 3 alkoxy, amino, Q-C 3 alkylamino, Q-C 3 dialkylamino, cyano and carboxy.
• R2a represents an optionally substituted group selected from CrC 4
• alkylcarbonyl C 1 -C 4 alkoxycarbonyl and phenylcarbonyl in said group of compounds, more preferably a CrC 4 alkylcarbonyl group which is unsubstituted or substituted with a group -NR11R10, wherein
• Rl l represents C 1 -C 4 alkyl or, more preferably, hydrogen and
• RIO represents hydrogen, Ci-C 4 alkyl, C 1 -C 4 alkylcarbonyl or phenylcarbonyl.
• Rl, R3 and R4 are preferably hydrogen, Q-C 3 alkyl, halogen, Q-C 3 alkoxy, amino, Q-C 3 alkylamino, Q-C3 dialkylamino or hydroxy in said group of compounds, most preferably hydrogen.
• R5 is CrC 4 alkyl in said compounds, preferably methyl, and R6, R7, R8 and R9 are selected from hydrogen, Q-C 3 alkyl, halogen, Q-C 3 alkoxy, amino, Q-C 3 alkylamino, C - C3 dialkylamino and hydroxyl, preferably from hydrogen, halogen, Q-C 3 alkoxy, and hydroxyl.
• R6 and R9 are hydrogen and R7 and R8 are independently hydrogen or methoxy.
• Al represents an optionally substituted group selected from aryl, heteroaryl, cycloalkyl and heterocyclyl;
• R2 represents a group -OR2a
• R2a represents hydrogen, formyl or an optionally substituted group selected from alkylcarbonyl, alkoxycarbonyl, arylcarbonyl and heteroarylcarbonyl;
• R2a represents hydrogen, Q-Csalkyl, formyl or an optionally substituted group selected from C 1 -C 4 alkylcarbonyl, C 1 -C 4 alkoxycarbonyl, Ce-Cioarylcarbonyl and
• Al represents an optionally substituted group selected from CrC 4 alkyl, C6-C 10 aryl, Cs-Cioheteroaryl, C3-C 7 cycloalkyl or Cs-Cvheterocyclyl; and A2 represents an optionally substituted group selected from C 1 -C 4 alkyl, C3-C 7 cycloalkyl, C 5 -C 7 heterocyclyl, -(CH 2 ) n -C6-C 1 oaryl and -(CH 2 ) n -C 5 -C 1 oheteroaryl;
• Rl, R3 and R4 represent hydrogen
• R5 represents methyl; - the compounds of formula (I), wherein
• R6, R7, R8 and R9 independently of one another represent:
• R6 and R9 both represent hydrogen and
• R7 and R8 independently of one another represent hydrogen or methoxy
• - Al represents an optionally substituted group selected from aryl, heteroaryl, cycloalkyl and heterocyclyl and
• R2 represents hydrogen or, in particular, a group -OR2a; wherein R2a represents methyl;
• Al represents an optionally substituted group selected from aryl and heteroaryl
• A2 represents an optionally substituted group selected from alkyl, -(CH 2 ) n -aryl and
• n 0 or 1, in particular, when
• Al is pyridyl or phenyl, optionally substituted with halogen or methoxy
• A2 is Ci-C 4 alkyl.
• R2 represents a group -OR2a
• R2a represents hydrogen, formyl or an optionally substituted group selected from alkylcarbonyl, alkoxycarbonyl, arylcarbonyl and heteroarylcarbonyl, in particular, when R2a is an optionally substituted group selected from C 1 -C 4 alkylcarbonyl,
• R2a is a C 1 -C 4 alkylcarbonyl group which is unsubstituted or substituted with a group -NR11R10, wherein Rl 1 is hydrogen or Ci-C 4 alkyl, in particular hydrogen, and
• RIO is hydrogen, CrC 4 alkyl, C 1 -C 4 alkylcarbonyl or phenylcarbonyl.
• R2 represents a group -OR2a
• R2a represents hydrogen, formyl or an optionally substituted group selected from alkylcarbonyl, alkoxycarbonyl, arylcarbonyl and heteroarylcarbonyl n represents 0 or 1 and G is a group -OC0 2 (CH 2 ) n -Al, wherein
• Al represents an optionally substituted group selected from Ci-C 4 alkyl, Ce-Cioaryl, Cs-Cioheteroaryl, C3-C 7 cycloalkyl or Cs-Cvheterocyclyl, in particular
• n 0 and Al is C 1 -C 4 alkyl.
• Particularly preferred are also compounds of formula (I) which have more than one of the features of the preferred embodiments of the compounds described above in combination.
• salts are especially the pharmaceutically acceptable salts of a compound of formula. I.
• Such salts are formed, for example, as acid addition salts, preferably with organic or inorganic acids.
• Suitable inorganic acids are, for example, halogen acids, such as hydrochloric acid, sulfuric acid, or phosphoric acid.
• Suitable organic acids are, for example, carboxylic, phosphonic, sulfonic or sulfamic acids, for example acetic acid, propionic acid, octanoic acid, decanoic acid, dodecanoic acid, glycolic acid, lactic acid, fumaric acid, succinic acid, adipic acid, pimelic acid, suberic acid, azelaic acid, malic acid, tartaric acid, citric acid, amino acids, such as glutamic acid or aspartic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, cyclohexanecarboxylic acid, adamantane carboxylic acid, benzoic acid, salicylic acid, 4-aminosalicylic acid
• 2-naphthalenesulfonic acid 1,5-naphthalene-disulfonic acid, 2-, 3- or 4-methyl- benzenesulfonic acid, methylsulfuric acid, ethylsulfuric acid, dodecylsulfuric acid, N-cyclohexylsulfamic acid, N-methyl-, N-ethyl- or N-propyl-sulfamic acid, or other organic protonic acids, such as ascorbic acid.
• compound of formula (I) is also meant herein to refer to hydrates and solvates, preferably pharmaceutically acceptable solvates, of these compounds.
• the compounds of formula (I) according to the present invention and salts, solvates or hydrates thereof can be prepared according to known methods, as described herein or variations thereof that will be apparent to those skilled in the art, followed, if necessary, by removing any protecting groups, forming a pharmaceutically acceptable salt or forming a pharmaceutically acceptable solvate or hydrate.
• R5 is not methyl
• Compounds in which R5 is not methyl can be prepared by methods as described in R. A. Lucas et al., J. Am. Chem. Soc. 1960, 82, 493-495; M. F. Bartlett, W. I. Taylor,
• compounds of formula (I ) can be obtained by a process in which a compound of formula ( ⁇ )
• the invention also relates to the use of a combination of a compound of formula (I) -with the exception of syrosingopine- and a mitochondrial inhibitor, e.g. metformin (and related biguanides, in particular phenformin and buformin), and to pharmaceutical products comprising a compound of formula (I) -with the exception of syrosingopine, in particular one of the preferred embodiments of compounds of formula (I) described above, and a mitochondrial inhibitor for use in the treatment of cancer, in particular for the treatment of carcinoma, leukemia, myeloma and lymphoma, and for achieving immunosuppression in autoimmunity, transplantation medicine and in other cases where immunosuppression is desirable, such as diseases of the skin, in particular psoriasis, nervous system, in particular multiple sclerosis, and of the haemopoietic system, in particular anemias; to the use of a combination of a compound of formula (I) and a mitochondrial inhibitor,
• metformin (and related biguanides like phenformin and buformin), for the preparation of a pharmaceutical composition for the treatment of cancer and achieving immunosuppression, and to methods of treatment of cancer and of achieving immunosuppression using a combination of a compound of formula (I) and a
• mitochondrial inhibitor e.g. metformin (and related biguanides like phenformin and buformin), or pharmaceutical compositions comprising a compound of formula (I) and a mitochondrial inhibitor.
• the invention relates furthermore to the use of a combination of a compound of formula (I) and a mitochondrial inhibitor, and of pharmaceutical compositions comprising a compound of formula (I) and a mitochondrial inhibitor for the treatment of cancer, in particular for the treatment of carcinoma, leukemia, myeloma, and lymphoma, and for the treatment of immunological disorders such as autoimmunity.
• Mitochondrial inhibitors as understood in the present invention comprise compounds which reduce mitochondrial activity and demonstrate varying degrees of
• Mitochondrial inhibitors comprise so-called uncoupling agents, which uncouple the flow of protons from ATP synthesis in mitochondria, and inhibitors that target different complexes of the electron transfer chain (ETC) in
• mitochondria e.g. complex I, complex II, complex ⁇ , complex IV, and complex V of the electron transfer chain.
• Further compounds considered to be mitochondrial inhibitors according to the invention are mitochondriotoxic compounds targeting the mitochondrial genome.
• Mitochondriotoxic drugs are also considered mitochondrial inhibitors according to the invention.
• Such mitochondriotoxic or mitochondrial inhibitory drugs synergize with a compound of formula (I) and represent anti-cancer agents when combined with a compound of formula (I).
• Mitochondriotoxic drugs have been used for treatment of very different clinical conditions (Cohen et al., Dev Disabil Res Rev 2010, 16: 189-199).
• Mitochondrial inhibitors according to the invention comprise:
• drugs used in liver or gallbladder disease with mitochondrial side effects such as tetracycline, ibuprofen, amiodarone, pirprofen, tamoxifen, valproate, chloroquine, quinidine, chlorpromazine, ketoconazole, cyclosporine A, rifampicine, and glyburine; inhibitors of electron transport chain complex I, such as amytal, capsaicin, haloperidol, risperidone, metformin, buformin, phenformin, bupivacaine, lidocaine, halothane, dantrolene, phenytoin, clofibrate, and fenofibrate;
• inhibitors of electron transport chain complex I such as amytal, capsaicin, haloperidol, risperidone, metformin, buformin, phenformin, bupivacaine, lidocaine, halothane,
• inhibitors of electron transport chain complex II such as cyclophosphamide and ketoconazole;
• inhibitors of electron transport chain complex ⁇ such as antimycin A, acetaminophen, isoflurane, and sevoflurane;
• inhibitors of electron transport chain complex IV such as cephaloridine, cefazolin, and cefalotin;
• inhibitors of electron transport chain complex V such as oligomycin
• inhibitors of mitochondrial DNA synthesis such as AZT (itovudidine), d4T (stavudine), ddl (didanosine), and ddC (zalcitabine);
• uncouplers of oxidative phosphorylation such as pentamidine, indomethacin, fluoxetine, propofol, aspirin, bubivacaine, tolcapone, and dinitrophenol;
• agents which reduce molecular oxygen to superoxide via a redox mechanism such as doxorubicin, isoniazid, gentamycin, and fluoroquinolone; and
• inhibitors of mitochondrial gene transcription such as interferon-alpha and interferon- gamma.
• Metformin is 3-(diaminomethylidene)-l,l-dimethylguanidine :
• Phenformin is l-(diaminomethylidene)-2-(2-phenylethyl)guanidine:
• metformin, phenformin and other mitochondrial inhibitors having basic nitrogen atoms mean the free base or any acid addition salt thereof.
• Such salts are formed, for example, as acid addition salts, preferably with organic or inorganic acids.
• Suitable inorganic acids are, for example, halogen acids, such as hydrochloric acid, sulfuric acid, or phosphoric acid.
• Suitable organic acids are, for example, carboxylic, phosphonic, sulfonic or sulfamic acids, for example acetic acid, propionic acid, octanoic acid, decanoic acid, dodecanoic acid, glycolic acid, lactic acid, fumaric acid, succinic acid, adipic acid, pimelic acid, suberic acid, azelaic acid, malic acid, tartaric acid, citric acid, amino acids, such as glutamic acid or aspartic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, cyclohexanecarboxylic acid, adamantane carboxylic acid, benzoic acid, salicylic acid, 4-aminosalicylic acid, phthalic acid, phenylacetic acid, mandelic acid, cinnamic acid, methane- or ethane- sulfonic acid, 2-hydroxyethanesulfonic acid, ethane- 1,2-dis
• 2-naphthalenesulfonic acid 1,5-naphthalene-disulfonic acid, 2-, 3- or 4-methyl- benzenesulfonic acid, methylsulfuric acid, ethylsulfuric acid, dodecylsulfuric acid, N-cyclohexylsulfamic acid, N-methyl-, N-ethyl- or N-propyl-sulfamic acid, or other organic protonic acids, such as ascorbic acid.
• the invention also relates to a pharmaceutical product comprising a compound of formula (I) as described above -with the exception of syrosingopine- and a mitochondrial inhibitor.
• Said pharmaceutical products may comprise one or more than one dosage unit comprising a compound of formula (I) and one or more than one dosage unit comprising a
• a pharmaceutical product according to the present invention can also comprise one or more than one dosage unit, wherein each of said dosage units comprises both, a compound of formula (I) and a mitochondrial inhibitor.
• Another embodiment of the invention is a pharmaceutical product which is free of mitochondrial inhibitors and which comprises a compound of formula (I) -with the exception of syrosingopine- and means for providing instructions for use of said pharmaceutical product, wherein said instructions for use include an instruction to use the pharmaceutical product in combination with a medicament comprising a mitochondrial inhibitor.
• Yet another embodiment of the invention is a pharmaceutical product comprising a mitochondrial inhibitor and means for providing instructions for use of said pharmaceutical product, wherein said instructions for use include an instruction to use the pharmaceutical product in combination with a medicament comprising a compound of formula (I) with the exception of syrosingopine.
• a specific embodiment of this product is free of compounds of formula (I).
• Means for providing instructions for the use of said product include, in particular, a package of the product and/or a package insert, on which the instructions are printed.
• Preferred are pharmaceutical products according to the invention comprising one or more than one dosage unit, wherein each of said dosage units comprises both, a compound of formula (I) and a mitochondrial inhibitor, i.e. fixed combinations of said components.
• compositions comprising a compound of formula (I) and a mitochondrial inhibitor and are, for example, compositions for enteral
• administration such as nasal, buccal, rectal or, especially, oral administration, and for parenteral administration, such as intravenous, intramuscular or subcutaneous
• compositions may comprise a compound of formula (I) and a mitochondrial inhibitor, e.g. metformin, alone or, preferably, together with a mitochondrial inhibitor, e.g. metformin, alone or, preferably, together with a mitochondrial inhibitor, e.g. metformin, alone or, preferably, together with a mitochondrial inhibitor, e.g. metformin, alone or, preferably, together with a mitochondrial inhibitor, e.g. metformin, alone or, preferably, together with a mitochondrial inhibitor, e.g. metformin, alone or, preferably, together with a mitochondrial inhibitor, e.g. metformin, alone or, preferably, together with a mitochondrial inhibitor, e.g. metformin, alone or, preferably, together with a mitochondrial inhibitor, e.g. metformin, alone or, preferably, together with a mitochondrial inhibitor, e.g. metformin, alone or, preferably, together with a mitochondrial inhibitor, e.g.
• the dosage of the combination of a compound of formula (I) and the mitochondrial inhibitor depends upon the disease to be treated and upon the species, its age, weight, and individual condition, the individual pharmacokinetic data, and the mode of administration.
• compositions comprise from approximately 1% to approximately 95% of the combination of a compound of formula (I) and a mitochondrial inhibitor, e.g. metformin, single-dose administration forms comprising in the preferred embodiment from approximately 20% to approximately 90% combination of a compound of formula (I) and a mitochondrial inhibitor, and forms that are not of single-dose type comprising in the preferred embodiment from approximately 5% to approximately 20% combination of a compound of formula (I) and mitochondrial inhibitor.
• Unit dose forms are, for example, coated and uncoated tablets, ampoules, vials, suppositories, or capsules. Further dosage forms are, for example, ointments, creams, pastes, foams, tinctures, drops, sprays, and dispersions. Examples are capsules containing from about 0.05 g to about 1.0 g
• compositions of the present invention are prepared in a manner known per se, for example by means of conventional mixing, granulating, coating, dissolving or lyophilizing processes.
• a compound of formula (I) and a mitochondrial inhibitor e.g. metformin
• suspensions or dispersions especially isotonic aqueous solutions, dispersions or suspensions which, for example in the case of lyophilized compositions comprising the combination of a compound of formula (I) and a mitochondrial inhibitor, alone or together with a carrier, for example mannitol, can be made up before use.
• compositions may be sterilized and/or may comprise excipients, for example preservatives, stabilizers, wetting agents and/or emulsifiers, solubilizers, salts for regulating osmotic pressure and/or buffers and are prepared in a manner known per se, for example by means of conventional dissolving and lyophilizing processes.
• excipients for example preservatives, stabilizers, wetting agents and/or emulsifiers, solubilizers, salts for regulating osmotic pressure and/or buffers and are prepared in a manner known per se, for example by means of conventional dissolving and lyophilizing processes.
• the said solutions or suspensions may comprise viscosity- increasing agents, typically sodium carboxymethylcellulose, carboxymethylcellulose, dextran, polyvinylpyrrolidone, or gelatins, or also solubilizers, e.g. Tween 80 ®
• Suspensions in oil comprise as the oil component the vegetable, synthetic, or semi- synthetic oils customary for injection purposes.
• liquid fatty acid esters that contain as the acid component a long-chained fatty acid having from 8 to 22, especially from 12 to 22, carbon atoms.
• the alcohol component of these fatty acid esters has a maximum of 6 carbon atoms and is a monovalent or polyvalent, for example a mono-, di- or trivalent, alcohol, especially glycol and glycerol.
• vegetable oils such as cottonseed oil, almond oil, olive oil, castor oil, sesame oil, soybean oil and groundnut oil are especially useful.
• injectable preparations are usually carried out under sterile conditions, as is the filling, for example, into ampoules or vials, and the sealing of the containers.
• Suitable carriers for preferred solid oral dosage forms are especially fillers, such as sugars, for example lactose, saccharose, mannitol or sorbitol, cellulose preparations, and/or calcium phosphates, for example tricalcium phosphate or calcium hydrogen phosphate, and also binders, such as starches, for example corn, wheat, rice or potato starch,
• disintegrators such as the above-mentioned starches, also carboxymethyl starch, crosslinked polyvinylpyrrolidone, alginic acid or a salt thereof, such as sodium alginate.
• Additional excipients are especially flow conditioners and lubricants, for example silicic acid, talc, stearic acid or salts thereof, such as magnesium or calcium stearate, and/or polyethylene glycol, or derivatives thereof.
• Tablet cores can be provided with suitable, optionally enteric, coatings through the use of, inter alia, concentrated sugar solutions which may comprise gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, or coating solutions in suitable organic solvents or solvent mixtures, or, for the preparation of enteric coatings, solutions of suitable cellulose preparations, such as acetylcellulose phthalate or hydroxypropyl- methylcellulose phthalate. Dyes or pigments may be added to the tablets or tablet coatings, for example for identification purposes or to indicate different doses of the combination of a compound of formula (I) and mitochondrial inhibitor.
• concentrated sugar solutions which may comprise gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide
• suitable organic solvents or solvent mixtures or, for the preparation of enteric coatings, solutions of suitable cellulose preparations, such as acetylcellulose phthalate or hydroxypropyl- methylcellulose phthalate.
• compositions for oral administration also include hard capsules consisting of gelatin, and also soft, sealed capsules consisting of gelatin and a plasticizer, such as glycerol or sorbitol.
• the hard capsules may contain the combination of a compound of formula (I) and mitochondrial inhibitor in the form of granules, for example in admixture with fillers, such as corn starch, binders, and/or glidants, such as talc or magnesium stearate, and optionally stabilizers.
• the combination of a compound of formula (I) and mitochondrial inhibitor is preferably dissolved or suspended in suitable liquid excipients, such as fatty oils, paraffin oil or liquid polyethylene glycols or fatty acid esters of ethylene or propylene glycol, to which stabilizers and detergents, for example of the polyoxyethylene sorbitan fatty acid ester type, may also be added.
• suitable liquid excipients such as fatty oils, paraffin oil or liquid polyethylene glycols or fatty acid esters of ethylene or propylene glycol, to which stabilizers and detergents, for example of the polyoxyethylene sorbitan fatty acid ester type, may also be added.
• suitable liquid excipients such as fatty oils, paraffin oil or liquid polyethylene glycols or fatty acid esters of ethylene or propylene glycol, to which stabilizers and detergents, for example of the polyoxyethylene sorbitan fatty acid ester type, may also be added.
• Pharmaceutical compositions suitable for rectal administration are, for example,
• mitochondrial inhibitor e.g. metformin
• a suppository base e.g. triglycerides, paraffin hydrocarbons, polyethylene glycols or higher alkanols.
• aqueous solutions of a combination of a compound of formula (I) and a mitochondrial inhibitor, or aqueous injection suspensions that contain viscosity-increasing substances, for example sodium carboxymethylcellulose, sorbitol and/or dextran, and, if desired, stabilizers, are especially suitable.
• the combination of a compound of formula (I) and mitochondrial inhibitor, optionally together with excipients, can also be in the form of a lyophilizate and can be made into a solution before parenteral administration by the addition of suitable solvents. Solutions such as are used, for example, for parenteral administration can also be employed as infusion solutions.
• Preferred preservatives are, for example, antioxidants, such as ascorbic acid, or
• microbicides such as sorbic acid or benzoic acid.
• a mitochondrial inhibitor e.g. metformin
• a compound of formula (I) e.g. metformin
• pharmaceutical compositions comprising a
• mitochondrial inhibitor and a compound of formula (I) according to the invention show therapeutic efficacy against different types of cancer including carcinomas, sarcomas, gliomas, leukemias, lymphomas, e.g. epithelial neoplasms, squamous cell neoplasms, basal cell neoplasms, transitional cell papillomas and carcinomas, adenomas and
• adenocarcinomas adnexal and skin appendage neoplasms, mucoepidermoid neoplasms, cystic neoplasms, mucinous and serous neoplasms, ductal-, lobular and medullary neoplasms, acinar cell neoplasms, complex epithelial neoplasms, specialized gonadal neoplasms, paragangliomas and glomus tumors, naevi and melanomas, soft tissue tumors including sarcomas, fibromatous neoplasms, myxomatous neoplasms, lipomatous neoplasms, myomatous neoplasms, complex mixed and stromal neoplasms, fibroepithelial neoplasms, synovial like neoplasms, mesothelial neoplasms, germ cell neoplasms,
• meningiomas nerve sheath tumors, granular cell tumors and alveolar soft part sarcomas, Hodgkkfs and non-Hodgkkfs lymphomas, other lymphoreticular neoplasms, plasma cell tumors, mast cell tumors, immunoproliferative diseases, leukemias including acute and chronic leukemias, miscellaneous myeloproliferative disorders, lymphoproliferative disorders and myelodysplasia syndromes.
• mitochondrial inhibitor and a compound of formula (I) according to the invention show also therapeutic efficacy against immunological diseases sensitive to blockade of T cell proliferation including connective tissue diseases such as lupus erythematodes,
• sclerodermia polymyositis/ dermatomyositis, mixed connective tissue disease, rheumatoid arthritis, Sjogren-syndrome, panarteriitis nodosa, Wegeners granulomatosis; systemic autoimmune diseases such as rheumatoid arthritis, Goodpasture's syndrome, Wegener's granulomatosis, polymyalgia rheumatica, Guillain-Barre syndrome, multiple sclerosis; localized autoimmune diseases such as type 1 diabetes mellitus, Hashimoto's thyroiditis, Graves' disease, celiac disease, Crohn's disease, ulcerative colitis, Addison's disease, primary biliary cirrhosis, autoimmune hepatitis, and giant cell arteritis.
• systemic autoimmune diseases such as rheumatoid arthritis, Goodpasture's syndrome, Wegener's granulomatosis, polymyalg
• the combination of the mitochondrial inhibitor and the compound of formula (I) according to the invention and a pharmaceutical compositions comprising a compound of formula (I) and a mitochondrial inhibitor, e.g. metformin, according to the invention may be applied in the form of fixed combinations.
• Such fixed combination may contain a compound of formula (I) and a mitochondrial inhibitor, e.g. metformin, in a relative amount (weight per weight) of between 1 to 10 and 1 to ⁇ 000, preferably between 1 to 100 and 1 to 200, such as a combination of 1 to 130, with the maximum recommended daily dose of metformin being based on the experience with its use in diabetes type 2 therapy.
• a covalent linkage between a compound of formula (I) and some of the mitochondrial inhibitors, e.g. metformin may be envisaged.
• the combination of a compound of formula (I) and a mitochondrial inhibitor, e.g. metformin may be applied in two different, separate pharmaceutical compositions, optionally being provided together in a kit.
• the administration of the compound of formula (I) and the mitochondrial inhibitor, e.g. metformin can be administered simultaneously or a compound of formula (I) is administered separately before or after the mitochondrial inhibitor.
• the compounds may be given independently of one another within a reasonable time window.
• a treatment of cancer or autoimmune diseases or in an immunosuppressive treatment with a separate medicament containing a compound of formula (I) in combination with another approved medicament containing a mitochondrial inhibitor, like e.g. a commercially available metformin-containing medicament for the treatment type-2 diabetes, is another specific embodiment of the present invention.
• compositions comprising a compound of formula (I) and a mitochondrial inhibitor, e.g. metformin, may be further combined with other chemotherapeutic agents.
• Therapeutic agents for possible combination are especially one or more cytostatic or cytotoxic compounds, for example a chemotherapeutic agent or several selected from the group comprising indarubicin, cytarabine, interferon, hydroxyurea, bisulfan, or an inhibitor of polyamine biosynthesis, an inhibitor of the mTOR pathway, an inhibitor of mTOR- complex 1 or mTOR complex 2, an inhibitor of protein kinase, especially of serine/threonine protein kinase, such as protein kinase C, or of tyrosine protein kinase, such as epidermal growth factor receptor tyrosine kinase, a cytokine, a negative growth regulator, such as TGF- ⁇ or IFN- ⁇ , an aromatase inhibitor, a classical cytostatic, an inhibitor of the interaction of
• metformin, and pharmaceutical compositions comprising a compound of formula (I) and mitochondrial inhibitors may be administered especially for cancer therapy in combination with radiotherapy, immunotherapy, surgical intervention, or a combination of these.
• Long- term therapy is equally possible as is adjuvant therapy in the context of other treatment strategies or neo-adjuvant therapy in combination with surgery.
• Other possible treatments are therapy to maintain the patient' s status after tumor regression, or chemopreventive therapy, for example in patients at risk.
• the present invention relates furthermore to a method for the treatment of cancer and of immunological disorders such as autoimmunity, which comprises administering a combination of a compound of formula (I) and a mitochondrial inhibitor, e.g. metformin, in a quantity effective against said disease, to a warm-blooded animal requiring such treatment.
• a compound of formula (I) and mitochondrial inhibitors e.g. metformin
• the combination of a compound of formula (I) and mitochondrial inhibitors, e.g. metformin can be administered as such or especially in the form of pharmaceutical compositions, prophylactically or therapeutically, preferably in an amount effective against the said diseases, to a warm-blooded animal, for example a human, requiring such treatment.
• the daily dose administered is from approximately 0.05 g to approximately 3 g, preferably from approximately 0.25 g to approximately 1.5 g, of a combination of the present invention.
• the invention also relates to the use of a combination of a compound of formula (I) and a mitochondrial inhibitor, e.g. metformin, and of pharmaceutical compositions comprising a compound of formula (I) and a mitochondrial inhibitor for the treatment of cancer, in particular for the treatment of the particular cancers mentioned above. More specifically, the invention relates to the use of a combination of a compound of formula (I) and a mitochondrial inhibitor and of pharmaceutical compositions comprising a compound of formula (I) and a mitochondrial inhibitor for the treatment of carcinomas, sarcomas, leukemias, myelomas, lymphomas, and cancers of the nervous system. Furthermore, the invention relates to the use of a combination of a compound of formula (I) and a mitochondrial inhibitor and of pharmaceutical compositions comprising a compound of formula (I) and a mitochondrial inhibitor for achieving immunosuppression in
• the invention relates to the use of a combination of a compound of formula (I) and a mitochondrial inhibitor, e.g. metformin, and of
• compositions comprising a compound of formula (I) and a mitochondrial inhibitor for the treatment of autoimmune diseases, such as autoimmune diseases of the skin, nervous system, connective tissue, muscle, nervous system, blood forming system, bone and inner organs, in particular psoriasis, multiple sclerosis, and anemias.
• autoimmune diseases such as autoimmune diseases of the skin, nervous system, connective tissue, muscle, nervous system, blood forming system, bone and inner organs, in particular psoriasis, multiple sclerosis, and anemias.
• the preferred relative amount of a compound of formula (I) and mitochondrial inhibitor, e.g. metformin, dose quantity and kind of pharmaceutical composition, which are to be used in each case, depend on the type of cancer or autoimmune disease, the severity and progress of the disease, and the particular condition of the patient to be treated, and has to be determined accordingly by the physician responsible for the treatment.
• the mitochondrial inhibitor is selected from metformin, buformin, phenformin, rotenone, piericidin A, epiberberine, 2-thenoyltrifluoroacetone (TTFA), antimycin A, carbonyl cyanide-/?-trifluoromethoxyphenylhydrazone (FCCP), and stavudine and the relative amount (weight per weight) of the compound of formula (I) and mitochondrial inhibitor is between 1 to 10 and 1 to 1000, preferably between 1 to 10 and 1 to 500, e.g. between 1 to 10 and 1 to 200;
• the mitochondrial inhibitor is metformin and the relative amount (weight per weight) of compound of formula (I) and metformin is between 1 to 10 and 1 to 200;
• the mitochondrial inhibitor is oligomycin and the relative amount (weight per weight) of compound of formula (I) and oligomycin is between 000 to 1 and 10 ⁇ 00 to 1.
• a further aspect of the present invention is a pharmaceutical product as described herein for use in the treatment of cancer or autoimmune diseases or in an immunosuppressive treatment.
• the invention further relates to the use of a combination of a compound of formula (I) - with the exception of syrosingopine- and a mitochondrial inhibitor, e.g. metformin, for the preparation of a pharmaceutical composition for the treatment of cancer or autoimmune disease, as explained above, as well as to the use of a -with the exception of
• syrosingopine for the manufacture of a medicament for use in the treatment of cancer or autoimmune diseases or in an immunosuppressive treatment in combination with a mitochondrial inhibitor or to the use of a mitochondrial inhibitor, e.g. metformin, for the manufacture of a medicament for use in the treatment of cancer or autoimmune diseases or in an immunosuppressive treatment in combination with a -with the exception of syrosingopine.
• the invention provides a method for treatment of cancer or autoimmune disease, which comprises administering a combination of a compound of formula (I) and a mitochondrial inhibitor, e.g. metformin, or of a pharmaceutical composition comprising a compound of formula (I) and a mitochondrial inhibitor, in a quantity effective against said disease, to a warm-blooded animal requiring such treatment.
• a mitochondrial inhibitor e.g. metformin
• a pharmaceutical composition comprising a compound of formula (I) and a mitochondrial inhibitor
• the invention further relates to a method for the determination whether a cancerous cell is responsive to a compound of formula (I) treatment comprising the steps of
• step (c) incubating the cancerous cell of step (b) with a positively charged fluorescent dye
• step (e) comparing the measured fluorescence intensity of step (d) with the measured fluorescence intensity of the cancerous cell incubated with the positively charged fluorescent dye alone,
• the cancerous cell is a cell isolated from a potential patient to be treated with a combination of a compound of formula (I) and a mitochondrial inhibitor.
• Suitable media for culturing such cancerous cells are well known in the art, and include, for example Iscoves modified Dulbecco medium (IMDM) or RPMI 1640 medium.
• IMDM Iscoves modified Dulbecco medium
• RPMI 1640 medium Prior to testing, a single cell suspension from the ex vivo tumor material has to be prepared. Again, suitable standardized commercial methodologies are at hand, where physical disruption and enzymatic digestion steps are combined (see for example the method by
• TMRE tetramethylrhodamine ethyl ester perchlorate
• Rhodamine 123 rhodamine methyl ester chloride
• Rhodamine B tetraethylrhodamine hydrochloride
• MitoTracker Red CMXRos® CAS designation 1H,5H,11H, 15H- xantheno[2,3,4-ij:5,6,7-i'j']diquinolizin- 18-ium, 9-[4-(chloromethyl)phenyl]- 2,3,6,7, 12, 13, 16, 17-octahydro-, chloride
• carbocyanines JC- 1 5,5',6,6'- tetrachloro- l,l',3,3'-tetraethylbenzimidazolocarbocyanine io
• TMRM tetramethylrhodamine methyl ester
• evaporations are carried out by rotary evaporation under reduced pressure and work-up procedures are carried out after removal of residual solids by filtration;
• each intermediate is purified to the standard required for the subsequent stage and is characterized in sufficient detail to confirm that the assigned structure is correct;
• Acetone- ⁇ i ⁇ 5 Deuterated acetone
• reaction mixture After agitation at this temperature for 30 min, the reaction mixture is diluted with 4 mL of methanol and concentrated under reduced pressure. The residue is purified by preparative HPLC (eluent: water with 0.1% formic acid and acetonitrile; gradient) to give 90 mg of the desired product as off-white solid (formic acid salt).
• reaction mixture is washed with 20 mL of water, dried over Na 2 S0 4 , filtered and evaporated under reduced pressure.
• residue is purified by preparative HPLC (eluent: water with 0.1% TFA and acetonitrile; gradient) to give 97 mg of the desired product as light yellow solid (TFA salt).
• reaction mixture After agitation at 20°C for 20 hours, the reaction mixture is diluted with 50 mL of ethyl acetate, washed three times with 20 mL of water, dried over Na 2 S0 4 , filtered and concentrated under reduced pressure. The residue is purified by preparative HPLC (eluent: water with 0.02% ammonia and acetonitrile; gradient) to give 30 mg of the desired product as white solid.
• the product is obtained according to Example 6 from 4-nitrophenyl[pyridine-3- yl]methyl]carbonate (CAS 32939-32-5) and methyl (lR,15S,17R,18R,19S,20S)- 17-[(4- hydroxy-3,5- dimethoxyphenyl)carbonyloxy]-6,18-dimethoxy-3, 13- diazapentacyclo[l 1.8.0.0 2 ' 10 .0 4 ' 9 .0 15 ' 20 ]henicosa-2(10),4,6,8-tetraene-19-carboxylate (CAS 21432-74-6).
• the product is obtained according to Example 6 from 4-nitrophenyl-(3,5- dimethoxybenzyl)carbonate (CAS 6453-62-9) and methyl (1R,15S, 17R, 18R, 19S,20S)-17- [(4-hydroxy-3,5- dimethoxyphenyl)carbonyloxy]-6,18-dimethoxy-3, 13- diazapentacyclo[11.8.0.0 2 ' 10 .0 4 ' 9 .0 15 ' 20 ]henicosa-2(10),4,6,8-tetraene-19-carboxylate (CAS 21432-74-6).
• the product is obtained according to Example 6 from 4-nitrophenyl-(4- chlorobenzyl)carbonate (CAS 97534-88-8) and methyl (lR, 15S,17R,18R,19S,20S)- 17-[(4- hydroxy-3,5- dimethoxyphenyl)carbonyloxy]-6,18-dimethoxy-3, 13- diazapentacyclo[11.8.0.0 2 ' 10 .0 4 ' 9 .0 15 ' 20 ]henicosa-2(10),4,6,8-tetraene-19-carboxylate (CAS 21432-74-6).
• the product is obtained according to Example 6 from 4-nitrophenyl-(3,5- dichlorobenzyl)carbonate (prepared according to the preparation of 4-nitrophenyl[pyridine- 4-yl]methyl]carbonate ) and methyl (lR, 15S,17R,18R, 19S,20S)- 17-[(4-hydroxy-3,5- dimethoxyphenyl)carbonyloxy] -6, 18-dimethoxy-3, 13- diazapentacyclo[l 1.8.0.0 2 ' 10 .0 4 ' 9 .0 15 ' 20 ]henicosa-2(10),4,6,8-tetraene-19-carboxylate (CAS 21432-74-6).
• the product is obtained according to Example 6 from 4-nitrophenyl-(4- methoxybenzyl)carbonate (CAS 25506-37-0) and methyl (1R,15S, 17R, 18R, 19S,20S)-17- [(4-hydroxy-3,5- dimethoxyphenyl)carbonyloxy]-6,18-dimethoxy-3, 13- diazapentacyclo[l 1.8.0.0 2 ' 10 .0 4 ' 9 .0 15 ' 20 ]henicosa-2(10),4,6,8-tetraene-19-carboxylate (CAS 21432-74-6).
• the residue is purified by MCI gel chromatography (eluent: water/acetonitrile/TFA 4/1/0.1 -1/2/0.1; v/v/v). After evaporation of the acetonitrile under reduced pressure, the aqueous phase is extracted with ethyl acetate, the organic phase is washed with brine, dried over magnesium sulfate, filtered and
• the reaction mixture is stirred for 3 h at room temperature, diluted with DCM and washed subsequently with water, 5% aqueous citric acid solution, 5% aqueous sodium bicarbonate solution and brine.
• the organic phase is dried over magnesium sulfate, filtered and concentrated under reduced pressure.
• the crude product is treated with a mixture of DCM, ethyl acetate and diisopropylether, and the formed precipitate is further purified by preparative HPLC to give 108 mg of the desired product as light yellow solid.
• the residue is purified by MCI gel chromatography (eluent: water/acetonitrile/TFA 4/1/0.1 -1/2/0.1; v/v/v). After evaporation of the acetonitrile under reduced pressure, the aqueous phase is extracted with ethyl acetate, the organic phase is washed with brine, dried over magnesium sulfate, filtered and
• the reaction mixture is stirred for 3 h at room temperature, diluted with DCM and washed subsequently with water, 5% aqueous citric acid solution, 5% aqueous sodium bicarbonate solution and brine.
• the organic phase is dried over magnesium sulfate, filtered and concentrated under reduced pressure.
• the crude product is treated with a mixture of DCM and diisopropylether, and the formed precipitate is filtered and dried under vacuum to give 26 mg of the desired product as light brown solid.
• Example 27 Example 29:
• Example 42 is prepared by using 3-[[(l,l-dimethylethyl)dimethylsilyl]oxy]- benzenemethanol (CAS 96013-77-3) and final cleavage of the silylether using potassium carbonate in DMF.
• Example 44 is prepared by using 3-nitro-benzenemethanol (CAS 619-25-0) and final Fe/FeS0 4 mediated reduction of the nitro-group Example 30:
• Example 34 Example 35:
• Example 40 Example 42:
• Oncogene 2011, 30: 1551-65) Cells are seeded into 96-well microtiter plates (seeding cell density is 7,000 cells per well in 150 uL Iscove's medium) and test compounds are added (compounds of formula (I) are added from a 10 mM stock solution in DMSO, metformin from a freshly prepared 1 M solution in culture medium). Plates are incubated for 3 days at 37°, 5% C0 2 and proliferation is determined by AlamarBlue staining (0.1vol added to cultures and incubated for further 4-5 hours). The conversion of AlamarBlue is
• the same assay conditions can be used for testing the anti-proliferative activity of compounds of formula (I) in combination with other inhibitors of mitochondrial function like rotenone, piericidin A, epiberberine, 2-thenoyltrifluoroacetone (TTFA), sodium malonate, antimycin A, KCN, sodium azide, oligomycin, carbonyl cyanide-/?- trifluoromethoxyphenylhydrazone (FCCP) or stavudine, each used at its appropriate concentration.
• rotenone piericidin A
• epiberberine epiberberine
• 2-thenoyltrifluoroacetone (TTFA) 2-thenoyltrifluoroacetone
• FCCP carbonyl cyanide-/?- trifluoromethoxyphenylhydrazone
• stavudine each used at its appropriate concentration.
• exemplified compounds of formula (I) at concentrations of 10 ⁇ and lower are combined with 4 mM of metformin, a strong anti-proliferative activity is observed.
• the Compound of Example 6-10, 12- 18, 20-21, 31-36, 38 of the present application e.g. exhibit an antiproliferative activity in combination with 4 mM metformin at concentrations of 5 ⁇ and lower.

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• 2013
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  • 2013-07-03 JP JP2015520908A patent/JP2015522048A/ja active Pending
  • 2013-07-03 WO PCT/EP2013/064048 patent/WO2014009222A1/fr not_active Ceased
  • 2013-07-03 US US14/413,122 patent/US9511064B2/en not_active Expired - Fee Related
• 2014
  • 2014-12-17 IL IL236321A patent/IL236321A0/en unknown

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